Heat shock transcription factor 1 (HSF1) is the principal regulator of the heat shock response, a highly conserved pathway that protects cells from a variety of stressors. HSF1 is a major target of sirtuin 1, a critical longevity factor. This proposal seeks to clarify how isoforms of mouse HSF1, designated 1 and 2, contribute to the heat shock response. Several key questions will be tested in this proposal. Can constitutively active versions of both isoforms be constructed? Are functional heterotrimers between HSF11 and 2 subunits formed? What is the profile of genes activated by HSF11 or 2? Are HSF1 isoforms predominantly nuclear proteins? The specific aims that will address these issues are: Aim 1 seeks to test the hypothesis that the mouse 2 isoform, like the 1 isoform, is an activator of heat shock proteins (hsps). Aim 2 will test the hypothesis that HSF11 and 2 isoforms have the potential to interact with each other in NIH 3T3 cells to form functional heterotrimers. The final Aim will examine the subcellular localization of HSF1 isoforms, to determine whether they are usually found in the nucleus or move there after heat shock. To carry out Aim 1, a series of site-directed mutations will be created in the mouse Hsf11 and 2 isoform genes to create distinct constitutively active forms of the protein. These altered forms of mouse HSF11 and 2 will be tested in transiently transfected NIH3T3 cells to determine whether they can activate target hsp genes in the absence of heat shock, using quantitative real-time RT-PCR assays. To accomplish Aim 2, immunoprecipitation and protein gel electrophoresis of transfected NIH 3T3 cell extracts will be used to determine whether epitope tagged versions of Hsf11 and 2 interact. The binding of different HSF1 trimers to heat shock element sequences (HSEs) of several hsps will then be assessed by chromatin immunoprecipitation. To investigate the final Aim, tagged constitutive or native isoforms of HSF1 will be detected in transiently transfected NIH 3T3 cells by immunofluorescence relative to a nuclear marker, under conditions with or without heat shock. These studies will provide a systematic comparison of mouse HSF1 isoforms, examining their respective roles in transcriptional activation of hsp genes, potential for interacting as heteromeric proteins, and subcellular localization. The need for more complete characterization of HSF1 isoform function is essential to developing and testing new treatments for age-related diseases that target HSF1.